DESCRIPTION: This proposal is aimed at understanding the mechanisms and processes that specify the development of Drosophila muscle. In both vertebrates and Drosophila, muscle cells are formed from mesodermally derived stem cells that give rise to committed myoblasts. In the embryo and adult, these myoblasts form the somatic and visceral muscles. Only two muscle gene systems, the tropomyosin I and II genes (TmI and TmII) and the beta-3-tubulin gene, have been studied in any detail in Drosophila. The TmI gene, which is the major focus of this proposal, is 5.5 kb long and encodes two muscle-type-specific tropomyosin isoforms by alternate splicing of one of the exons. The nearby TmII gene is 33 kb long and encodes three muscle and one cytoskeletal tropomyosin isoform by multiple alternative splicing. The muscle-specific expression of both genes is controlled by a pair of enhancers located within the first intron. Detailed analyses conducted in Dr. Storti's laboratory have dissected the enhancers of the TmI gene. The 5' most component of each enhancer contains the muscle activator (MA) region, which can activate transcription at the correct time and in the correct tissue but only at a basal level. Oligos from this region have been used to screen expression libraries for proteins that bind to the enhancer; to date, five proteins have been identified. Another protein, the Drosophila homolog of vertebrate MEF2, binds to a site downstream of the MA as well as to a site in the distal enhancer. This protein may function as a positive regulator of TmI transcription and may act in conjunction with the MA to facilitate high level expression in all embryonic, larval and adult somatic muscles. A different protein, CF2, also appears to play a role in regulating TmI transcription, possibly by acting in concert with D-mef2. The results of Dr. Storti's studies have therefore allowed him to develop a working model of how TmI (and possibly other muscle genes) is regulated. The project has two broad goals. One is to delineate more clearly the cis-elements regulating TmI expression and to determine if these elements act positively or negatively. In addition, the factors that bind to these cis-elements will be identified and studied. The second goal is to determine if and how the factors regulating TmI expression are involved in other aspects of myogenesis, such as commitment of mesodermal cells into the myogenic lineage and their subsequent differentiation. Many of the experiments proposed in the project are a continuation of ongoing work.